In conventional ion cyclotron resonance (“ICR”) mass spectrometers, such as those typically used in connection with Fourier Transform Mass Spectrometry (“FTMS”), charged particles are directed into a magnetic field such that the mass to charge ratio (M/Z) of the particles can be measured. In one application of this technology, as described in U.S. Pat. No. 4,959,543, which is incorporated by reference herein in its entirety, charged particles are subjected to a high voltage pulse and caused to be accelerated to larger radii of gyration relative to the particles' natural radii of gyration. Once excited in this fashion, the charged particles move in circular orbits at frequencies given by the cyclotron equation, ω=B/(M/Z) (where B is the magnetic field strength and ω is the angular frequency). The excited cyclotron motions induce transient signals on a pair of parallel electrodes positioned inside the magnet; the transient signals are a measure of the cyclotron frequency of the particles. In fact, the transient signals are actually a composite of the cyclotron frequencies of all of the ions present in the magnet. By implementing certain Fourier transform mathematics (e.g., a Fast Fourier Transform, or “FFT,” algorithm to extract the frequency and amplitude for each frequency component), these transient signals are converted into a frequency spectrum (i.e., frequency peaks corresponding to each ionic species in the instrument). In this first order model, measured frequencies are converted into M/Z through calibration values when the magnetic field strength (B) is known. There are a number of commercially available products that implement the FTMS technique; for example, Thermo, Bruker, and IonSpec all produce FTMS instruments that generally function in this manner.
As noted above, FTMS exploits the property that an ion of mass M and charge Z placed in a magnetic field of strength B undergoes orbital motion with angular frequency B/(M/Z). In a mass spectrometer, ions must be trapped by an external electrostatic field producing a slight shift in the cyclotron frequency given above. Additional frequency shifts are produced by the electrostatic field produced by the population of ions in the instrument, known as the “space-charge effect” (Gorshov. et al., Amer. Society Mass Spectrom. 4:855-868, 1991). Variations in the frequency observed for a particular ion (with fixed M/Z) can be due to fluctuations in the strength of the magnetic field, trapping voltage, or the “space-charge” effect. Of these three factors, the space-charge effect is believed to be the most difficult to control and to model. Variations in the space-charge effect are significant in liquid-chromatography mass spectrometry (LCMS), the standard technique used in analysis of proteomic samples. These variations are best corrected by active real-time calibration.
Efforts to extract accurate mass information from FTMS by mass calibration have been previously investigated. See L. K. Zhang et al., Mass Spectrometry Reviews, 24:286-309 (2005). Previous methods of FTMS mass calibration include the use of “internal” calibrants, and/or the use of “external” calibrants. In external, or “off-line” calibration, a set of standard molecules of known mass are measured by the instrument separately from the experimental sample. The differences between the measured and true masses are known with certainty, and the calibration parameters are adjusted to minimize these differences. The primary limitation of external calibration is that the calibration parameters do not remain constant from one scan to the next, largely due to the space charge effect. See E. B. Ledford, Jr. et al., Anal. Chem., 56:2744-2748 (1984).
Internal or “on-line” calibration involves the infusion of standard molecules of known mass into an experimental sample, or directly into the mass spectrometer in parallel with the sample, and measuring the mass of the standards and experimental sample in the same scan. However, the signal from the calibrant molecules may obscure a signal arising from the sample through “ion suppression”. Ion suppression occurs because the total ion capacity of an FTMS instrument is generally fixed. Therefore, the calibrant molecules are analyzed at the expense of analyte ions, reducing the measured analyte signal.
A number of methods have attempted to perform calibration without added calibrants in a process called “direct calibration”. One approach (described in M. Mann, Proceedings of the 43rd ASMS Conference on Mass Spectrometry and Allied Topics, Atlanta, 1995) is based upon Mann's insight that peptide masses are confined to clusters of values spaced roughly 1 Dalton (10-100 ppm) apart throughout the spectrum (Wool et al., Proteomics, 2:1365-1373, 2002). While this method may be useful for low mass accuracy mass spectrometers (e.g., MALDI-TOF), it is not suitable for use with higher mass-accuracy systems such as FTMS. In these methods, peptides are either matched to a distribution (not identified) or only peptides that are known to be in the sample a priori are identified.
Another direct calibration method uses the known mass spacings between different charge states of the same molecule as calibration constraints (Bruce et al., JASMS 11:416-421, 2000). However, this method is unable to match the accuracy of FTMS frequency measurements. Yanofsky et al. disclose a method for an internal recalibration of an FTICR-MS analysis (Anal. Chem 77:7246-7254, 2005). However, this method is a limited approach that uses the knowledge of a particular class of proteins, and requires partial knowledge of the sample components. Direct calibration methods have also been used to identify components in wine (Cooper, H. J., and Marshall, A. G., J. Agric. Food Chem, 49:5710-5718), and petroleum products (Marshall A. G. et al., Acc. Chem. Res. 37:53-59, 2004). These methods, however, also require a priori knowledge of the masses of some of the species in the sample.
There is a need in the art for improved calibration and peptide identification techniques in connection with mass spectrometry that obviate at least some of the aforementioned limitations of currently available technology.